One of the principal mechanisms by which cellular regulation is effected is through the transduction of extracellular signals into intracellular signals that in turn modulate biochemical pathways. Examples of such extracellular signaling molecules include growth factors, cytokines, and chemokines. The cell surface receptors of these molecules and their associated signal transduction pathways are therefore among the principal means by which cellular behavior is regulated. Because cellular phenotypes are largely influenced by the activity of these pathways, it is currently believed that a number of disease states and/or disorders are a result of either aberrant expression or functional mutations in the molecular components of signal transduction pathways. Consequently, considerable attention has been devoted to the characterization of these proteins.
Tumor necrosis factor (TNF), a cytokine produced by activated macrophages, initiates a variety of cellular responses. These responses include proliferation, differentiation and activation as well as apoptosis, inflammatory and immunoregulatory responses, and all are mediated through one of two receptors, TNF-R55 or TNF-R75 (Adam-Klages et al., Cell, 1996, 86, 937-947).
Unlike most receptors, TNF-R55 has no intrinsic enzymatic activity and therefore relays signals through protein binding motifs on its cytoplasmic domain. One such domain is known as the death domain and regulates binding of proteins involved in the signaling pathway leading to apoptosis. Another recently discovered binding domain is the the NSD (N-SMase activating domain). This domain comprises the binding site for a newly discovered protein, FAN.
FAN (factor associated with N-SMase activation), a regulatory protein of the WD-repeat protein family, was identified as a mediator of TNF-induced activation of N-SMase and likely plays a role in the inflammation process (Adam et al., J. Inflamm., 1995, 47, 61-66; Adam-Klages et al., Cell, 1996, 86, 937-947). Binding of TNF to the extracellular side of the receptor initiates a pathway on the intracellular side in which FAN activates the enzyme N-SMase (neutral sphingomyelinase). This enzyme, in turn, hydrolyzes sphingomyelin to release ceramide, an important second messenger, which then accumulates at the plasma membrane. This accumulation activates proline-directed protein kinases, which then activate the cytosolic phospholipase A2 producing arachidonic acid resulting in the generation of proinflammatory metabolites such as prostaglandins and leukotrienes (Adam-Klages et al., J. Leukoc. Biol., 1998, 63, 678-682).
Currently, there are no known therapeutic agents which effectively inhibit the synthesis of FAN. To date, strategies aimed at inhibiting FAN function have involved the use of peptide-hydroxamate metalloproteinase inhibitors to block the proteolytic processing of TNF-R55 and TNF-R75(Gallea-Robache et al., Cytokine, 1997, 9, 340-346). However, these targeting strategies are not specific to FAN and could affect all of the signaling pathways downstream of the TNF-R55 receptor. Consequently, there remains a long felt need for additional agents capable of effectively inhibiting FAN function.
Antisense oligonucleotides capable of inhibiting protein expression or function may prove useful in a number of therapeutic and research applications and could thereby provide a promising new pharmaceutical tool for the effective modification of the expression of specific genes.